Apatite pretreatment

ABSTRACT

Apatite pretreatment methods are provided. The method is applied to the apatite solid surface prior to first chromatographic use. In one embodiment, the method may be achieved by contacting an apatite solid surface with a phosphate buffered solution at a pH of at least about 6.5 and contacting the apatite solid surface with a solution having a hydroxide.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation application of U.S. patentapplication Ser. No. 14/747,181, filed Jun. 23, 2015, which claimspriority to U.S. Provisional Application No. 62/015,894, filed Jun. 23,2014; U.S. Provisional Application No. 62/082,017, filed on Nov. 19,2014; and U.S. Provisional Application No. 62/151,882, filed Apr. 23,2015, each of which is incorporated in its entirety herein for allpurposes.

BACKGROUND

Apatite solid support surfaces, including hydroxyapatite, ceramicapatite, fluorapatite, and fluoride enhanced apatite, among otherapatite solid surfaces, are used for purification of a wide variety oftarget analytes. Apatite is most commonly utilized for purification ofbiological analytes, including proteins, carbohydrates, polynucleotides,and viral particles. Apatite possesses unique properties as apurification support because it provides affinity, ion exchange and/orhydrophobic interaction modalities in a single support.

Apatite restoration methods restore loss of mass after an apatitepurification procedure. Loss of mass can, however, occur prior to anapatite purification procedure, i.e., during hydration of the dryapatite resin, column packing and prior to loading of the sample.

SUMMARY

Disclosed herein are apatite pretreatment methods. Also disclosed hereinare apatite regeneration methods.

In an embodiment, a method of treating an apatite solid surface prior touse comprises (a) contacting the apatite solid surface with a phosphatebuffered solution at a pH of at least about 6.5; and (b) contacting theapatite solid surface with a solution having a hydroxide. In someembodiments, step (a) is performed before step (b). In some embodiments,the phosphate buffered solution is a solution having from about 0.1 M toabout 1.0 M phosphate at a pH of from about 6.5 to about 10.0. In someembodiments, the phosphate buffered solution is 400 mM phosphate at a pHof 8.0. In certain embodiments, the hydroxide is an alkaline hydroxide.In some embodiments, the alkaline hydroxide is sodium or potassiumhydroxide.

In some embodiments, the method further includes purifying a targetanalyte with an apatite solid surface, the purifying comprising: (a)contacting the apatite solid surface with the target analyte, therebyseparating the target analyte from one or more contaminants; (b)collecting the target analyte; and (c) regenerating the apatite solidsurface the regenerating comprising, (i) contacting the apatite solidsurface with a buffered calcium solution comprising a calcium ion at aconcentration of at least about 10 mM and a zwitterionic buffer, whereinthe ratio of zwitterionic buffer concentration to calcium ionconcentration is at least about 2 and the pH of the solution is at leastabout 6.5; (ii) contacting the apatite solid surface with a phosphatebuffered solution at a pH of at least about 6.5; and (iii) contactingthe apatite solid surface with a solution comprising an hydroxide.

In one embodiment, the present invention provides a method wherein (a)comprises binding the target analyte to the apatite solid surface, and(b) comprises eluting the target analyte from the apatite solid surface.In another embodiment, (a) comprises contacting the apatite solidsurface to the target analyte, thereby flowing the target analytethrough the apatite solid surface, and (b) comprises collecting thetarget analyte in the flow through.

In one embodiment, the zwitterionic buffer is a sulfonic acid containingbuffer. In some cases, the sulfonic acid containing buffer is IVIES,PIPES, ACES, MOPSO, MOPS, BES, TES, HEPES, DIPSO, TAPS, TAPSO, POPSO, orHEPPSO, EPPS, CAPS, CAPSO, or CHES. In some cases, the sulfonic acidcontaining buffer is IVIES.

In one embodiment, the calcium ion is at least 1 mM, 2 mM, 3 mM, 4 mM, 5mM, 10 mM (e.g., 10.1 mM, 10.2 mM, 10.3 mM, 10.4 mM, or 10.5 mM), 20 mM,25 mM or at least about 50 mM. In another embodiment, the ratio ofzwitterionic buffer concentration to calcium ion concentration is atleast about 2.5, 3, or 4. In yet another embodiment, the bufferedcalcium solution comprises calcium chloride or calcium nitrate. In yetanother embodiment, the phosphate buffered solution comprises a solutioncontaining from about 0.1 M or 0.2 M to about 1.0 M phosphate or fromabout 0.1 M or 0.2 M to about 0.5 M phosphate, at a pH of from about 6.5to about 8. In some cases, the phosphate buffered solution comprises 400mM phosphate at a pH of 7.0.

In one embodiment, the hydroxide comprises an alkaline hydroxide. Insome cases, the alkaline hydroxide comprises sodium or potassiumhydroxide. In one embodiment, the regenerating reverses or eliminatesdegradation of the column that occurs during protein purification orcolumn cleaning steps. In another embodiment, the regenerating increasesthe strength of the apatite solid surface by at least about 1%, 5%, 10%,15%, 20%, or more.

In one embodiment the regenerating is performed before, or replaces, aphosphate cleaning/stripping step that elutes adsorbed biologicalcompounds. In some cases, the regenerating step is performed afterelution of target analyte.

In one embodiment, the step (ii) of contacting the apatite solid surfacewith a solution comprising phosphate at a pH of at least about 6.5further comprises: contacting the apatite solid surface with a solutioncomprising phosphate at a concentration of 10 mM, or less than about 10mM, at a pH of at least about 6.5 or 7; and then contacting the apatitesolid surface with a solution comprising phosphate at a concentration ofat least about 100 mM, 200 mM, 400 mM, or 500 mM, at a pH of at leastabout 6.5 or 7.

In one embodiment, the regenerating consists of (i), a wash, (ii), and(iii).

DETAILED DESCRIPTION

Described herein are methods for pretreating apatite solid surfaces.Pretreatment methods have been discovered that prevent or reduce thedeterioration (i.e., mass loss) of an apatite solid surface prior tofirst use in a chromatographic procedure for purifying a target moleculefrom a sample. A phosphate buffered solution followed by an alkalinehydroxide can be applied prior to a bind and elute or flow throughpurification procedure.

Also described herein are methods for regenerating apatite solidsurfaces. Regeneration methods have been discovered that reduce,eliminate, or reverse the deterioration of an apatite solid surfaceafter use in at least one chromatographic procedure by treating theapatite solid surface with a buffered calcium solution, followed by aphosphate buffered solution, followed by an alkaline hydroxide. Thebuffered calcium solution, phosphate buffered solution, and alkalinehydroxide can be applied subsequent to a bind and elute or flow throughpurification procedure.

Definitions

“Apatite” refers to a mineral of phosphate and calcium of the generalformula Ca₅(PO₄)₃(X), wherein X is a negatively charged ion. Generally,X is F, Cl, or OH. However, the structure and chemistry of apatite allowfor numerous substitutions, including a variety of metal cations (e.g.,one or more of K, Na, Mn, Ni, Cu, Co, Zn, Sr, Ba, Pb, Cd, Sb, Y, U, orvarious rare earth elements) that substitute for Ca in the structure,and anionic complexes (e.g., AsO₄ ⁻³, SO₄ ⁻², CO₃ ⁻², SiO₄ ⁻⁴, etc.)that substitute for PO₄ ⁻³.

“Hydroxyapatite” refers to a mixed mode solid support comprising aninsoluble hydroxylated mineral of calcium phosphate with the structuralformula Ca₁₀(PO₄)₆(OH)₂. Its dominant modes of interaction arephosphoryl cation exchange and calcium metal affinity. Hydroxapatite iscommercially available in various forms, including but not limited toceramic, crystalline and composite forms. Composite forms containhydroxyapatite microcrystals entrapped within the pores of agarose orother beads.

“Fluorapatite” refers to a mixed mode support comprising an insolublefluoridated mineral of calcium phosphate with the structural formulaCa₁₀(PO₄)₆F₂. Its dominant modes of interaction are phosphoryl cationexchange and calcium metal affinity. Fluorapatite is commerciallyavailable in various forms, including but not limited to ceramic andcrystalline composite forms.

An “apatite solid surface” refers to fused nanocrystals (ceramicapatite), microcrystals, or compounded microcrystals of apatite. Apatitesolid surfaces include, but are not limited to, hydroxyapatite, orfluorapatite. Ceramic apatites include, but are not limited to, ceramichydroxyapatite (e.g., CHT™) or ceramic fluorapatite. Ceramic apatitesare a form of apatite minerals in which nanocrystals are agglomeratedinto particles and fused at high temperature to create stable ceramicmicrospheres suitable for chromatography applications. Compoundedmicrocrystals include but are not limited to HA Ultragel® (Pall Corp.).Microcrystals include but are not limited to Bio-Gel HTP, Bio-Gel® HT,DNA-Grade HT (Bio-Rad) and Hypatite C (Clarkson Chromatography).

“Sample” refers to any composition having a target molecule or particleof interest. A sample can be unpurified or partially purified. Samplescan include samples of biological origin, including but not limited toblood, or blood parts (including but not limited to serum), urine,saliva, feces, as well as tissues. Samples can be derived fromunpurified, partially purified, or purified cell lysate or spent cellgrowth media.

“Target molecule” or “target analyte” refers to a molecule or analyte tobe detected in a sample. In some embodiments, the target molecule is apeptide, protein (e.g., an antibody, enzyme, growth regulator, clottingfactor, or phosphoprotein), polynucleotide (e.g., DNA, such as dsDNA orssDNA; RNA, such as mRNA or miRNA; or a DNA-RNA hybrid), aptamer,affimer, peptide nucleic acid, carbohydrate, virus, virus-like particle,drug compound, metabolite, or cell.

Deterioration of a resin that occurs during resin hydration and columnpacking can cause a loss in calcium, a loss in resin particle strengthand an increase in particle breakage. In some embodiments, such effectscan be prevented or reduced by the methods described herein.

Deterioration of a resin that occurs upon use in a chromatographicprocedure can cause the resin particles to lose their strength and thusto break apart into smaller particles causing blockage in the column.The deterioration can occur as a chemical breakdown of the apatite,causing a loss of mass which can in turn result in a loss of columnvolume, a loss in particle strength, an increase in particle breakage,or a combination thereof. In some embodiments, such effects can bereversed by the methods described herein. The reversal of deteriorationthat can be achieved by the practice of the methods described herein canresult in a lower rate of resin mass loss, a lower rate of decline inparticle strength, or both. In many cases, the reversal of deteriorationcan be accompanied by increases in resin mass, particle strength, orboth.

Mass of the apatite solid surface can be assayed by, e.g., weighing adried apatite sample, for example after washing away buffer componentsand adsorbed biological compounds. Apatite media strength can be assayedby, e.g., measuring resistance to agitational force (e.g., stirring),resistance to sonication, or resistance to compression (e.g.,application of a uniaxial compressive force). Resistance to sonicationor agitational force can be measured by inspection of the apatite solidsurface after the treatment to measure the generation of fines.Resistance to compression can be measured by measuring the forcerequired to compress a given mass of apatite to a constant terminalforce setting and determining the compressed distance. Apatitedeterioration or degradation can be measured relative to a sample thathas not been subjected to an apatite purification (i.e., purification ofa target molecule using apatite) or an apatite regeneration procedure.

An “alkaline hydroxide” refers to a metal alkali hydroxide comprisingany cation elements in Group I of the periodic table, including, e.g.,lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs),and francium (Fr). Thus, exemplary alkaline hydroxides include, forexample, NaOH, LiOH, and KOH.

A zwitterionic buffer is a buffer that can contain a formal positive anda formal negative electrical charge at the same time. Exemplaryzwitterionic buffers can include, but are not limited to, bufferscontaining a sulfonic acid group. As used herein, a “sulfonic acid”refers to a member of the class of organosulfur compounds with thegeneral formula RS(═O)₂—OH, where R is an organic group (e.g., alkyl, oralkene, or aryl) and the S(═O)₂—OH group is a sulfonyl hydroxide.

Exemplary zwitterionic buffers containing a sulfonic acid group caninclude, but are not limited to, aminoalkanesulfonic acids. Exemplaryaminoalkanesulfonic acids can include, but are not limited to,aminoalkanesulfonic acids with a minimum of two carbons between amineand sulfonic acid groups. Exemplary zwitterionic buffers containing asulfonic acid group can include, but are not limited to,N,N-dialkylaminomethanesulfonic acids.

Exemplary zwitterionic buffers containing a sulfonic acid group caninclude, but are not limited to, IVIES (2-(N-morpholino)ethanesulfonicacid), PIPES (1,4-Piperazinediethanesulfonic acid), ACES(2-(carbamoylmethylamino)ethanesulfonic acid), MOPSO(3-morpholino-2-hydroxypropanesulfonic acid), MOPS(3-morpholinopropane-1-sulfonic acid), BES(N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), TES(2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonicacid), HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid),DIPSO (3-(N,N-Bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid),TAPS(3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonicacid), TAPSO(3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonicacid), POPSO (piperazine-1,4-bis(2-hydroxypropanesulfonic acid)), orHEPPSO (N-(2-Hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonicacid)), EPPS (N-(2-Hydroxyethyl)piperazine-N′-(3-propanesulfonic acid)),CAPS (3-(Cyclohexylamino)-1-propanesulfonic acid), CAPSO(N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid), CHES(2-(Cyclohexylamino)ethanesulfonic acid), MOBS(4-(N-morpholino)butanesulfonic acid), TABS(N-tris(hydroxymethyl)-4-aminobutanesulfonic acid), or AMPSO(N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid).

Calcium ion for use as a restoration material in the proceduresdescribed herein can be supplied by calcium hydroxide or by a solublecalcium salt, typically a salt that is soluble in water. Calcium halidesand calcium nitrate are examples of calcium salts that can be used. Anexemplary calcium halide is calcium chloride. In some cases, calciumchloride is preferred when the chromatography buffer during flow throughpurification, or the elution buffer during bind and elute purification,contains an alkali metal chloride.

As used herein, the terms “buffer,” “buffered,” and the like, in thecontext of a buffered calcium solution refers to a buffer that iscompatible with (e.g., does not substantially interact with orprecipitate in complex with) calcium and is employed for the purpose ofstabilizing the pH of an aqueous solution at or near a specified value,or within a specified range. As such, generally, the “buffer” in abuffered calcium solution cannot be water. In some embodiments, the“buffer” in a buffered calcium solution is not phosphate.

Phosphate can be used in a variety of buffers for apatite equilibration,chromatography, elution, cleaning/stripping, or apatite regeneration.Phosphate can be supplied from any soluble phosphate salt, typically asalt that is soluble in water. Alkali metal or alkaline earth metalphosphates are examples, with sodium or potassium phosphate asparticularly convenient examples. Alkali or alkaline earth metalphosphate salts can be utilized in mono- and di-basic forms, or acombination thereof.

As used herein, the term “about” refers to the recited number and anyvalue within 10% of the recited number. Thus, “about 5” refers to anyvalue between 4.5 and 5.5, including 4.5 and 5.5.

I. Introduction

The presence of an ionic species in the buffer that is common to acomponent of the apatite solid surface (a common ion) can suppressleaching of that component from the apatite solid surface. Thus, calciumand/or phosphate buffers are often preferred during apatiteequilibration (e.g., hydration and column packing), loading, flowthrough, elution, or cleaning/stripping. Accumulation of hydronium ionson the apatite surface can occur due to a variety of mechanisms duringequilibration, loading, flow through, and washing steps. In particular,the presence of alkali metal salts can increase, or promote,accumulation of hydronium ions. A high pH phosphate solution (e.g.,phosphate at a pH of about 6.5 or higher) of sufficient concentration(e.g., 100, 200, 300, 400 mM, or higher), can provide buffering capacityto mitigate the pH shift that commonly occurs during hydronium ionrelease, and therefore reduce acid solubilization of the apatite. Theuse of alkali metal salts concurrently with a phosphate buffer of asuitable pH and concentration generally mitigates mass loss to asignificant degree. However, media strength can still be significantlydecreased. Neutralization of accumulated hydronium ions can reduce theamount of accumulated hydronium ions, and thus reduce degradation duringa subsequent phosphate buffer cleaning step.

Applicants have discovered that an apatite solid surface can bepretreated before use in a chromatographic procedure by treating with abuffered phosphate solution. After contacting the phosphate bufferedsolution with the apatite solid surface, the apatite solid surface canbe further treated with a hydroxide. The methods described hereinprovide a substantial and surprising degree of pretreatment. Thissubstantial and surprising degree of pretreatment can be indicated as areduction or elimination of degradation, as measured by loss in calcium.

Applicants have further discovered that the pretreatment methoddescribed herein can be combined with a regeneration method. Afterpretreatment, the apatite solid surface can be significantly regeneratedafter use in a chromatographic procedure by treating with a bufferedcalcium solution. In some embodiments, the regeneration method isapplied to the apatite solid surface intermittently, i.e., after thefirst chromatographic procedure and then after two, three or morechromatographic procedures. In some embodiments, the regeneration methodis applied to the apatite solid surface after each chromatographicprocedure. Generally, the buffered calcium solution is applied after thetarget molecule has been purified and collected. In some cases, thebuffered calcium solution is applied after the apatite solid surface hasbeen cleaned/stripped (e.g., with a high molarity phosphate buffer, suchas 400 mM phosphate) and/or sanitized. The buffered calcium solution canbe optionally washed away, and then the apatite treated with a phosphatebuffered solution. After contacting the phosphate buffered solution withthe apatite solid surface, the apatite solid surface can be furthertreated with a hydroxide. The regeneration procedures described hereinprovide a substantial and surprising degree of regeneration. Thissubstantial and surprising degree of regeneration can be indicated as areduction, elimination, or reversal of degradation, as measured bychange in apatite mass or loss in apatite strength.

II. Pretreatment Method

Described herein are apatite pretreatment methods for reducing orpreventing apatite deterioration by pretreating the apatite solidsurface with a phosphate buffered solution, followed by an alkalinehydroxide. The phosphate buffered solution and alkaline hydroxide areapplied prior to a protein purification procedure.

A. Phosphate Buffered Solution

The pretreatment method begins by contacting the apatite solid surfacewith a phosphate containing buffer. The phosphate concentration of thephosphate containing buffer and the amount of the phosphate containingbuffer passed through the resin can vary, but will generally be selectedas any amount that will prevent or reduce the deterioration of the resinthat occurs prior to apatite use (e.g., prior to purification). Withoutwishing to be bound by theory, it is believed that the phosphatecontaining buffer interacts with the apatite solid surface to generate aloosely bound (e.g., non-covalent) phosphate layer on the apatite solidsurface. Thus, an amount, volume, concentration, etc. of phosphate, orany other component or aspect of the phosphate containing buffer thatwill prevent or reduce the deterioration of the resin that occurs priorto a purification procedure can be an amount that allows for sufficientformation of a loosely bound phosphate layer.

The phosphate concentration of the phosphate containing buffer isgenerally selected to be below the solubility limit of the phosphate atthe pH and temperature of the buffer. Moreover, the concentration canvary based on presence or absence of other components of the buffer, orthe selected composition of any preceding buffer. In certain embodimentsof the concepts herein, best results will be achieved with a phosphateconcentration of from about 10 mM to about 1, 1.5, or 2 M; from about 20mM to about 1.5 M; or from about 25 mM to about 1 M; from about 50 mM toabout 1 M; including at least about, or about, 10 mM, 15 mM, 20 mM, 25mM, 30 mM, 40 mM, 50 mM, 60 ppm, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM,150 mM, 200 mM, 300 mM, 500 mM, 750 mM, 1 M, or higher. In some cases,the phosphate concentration is 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM,50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 150 mM, 200 mM, 300mM, 500 mM, 750 mM, 1 M, or higher. In some cases, the column iscontacted with a low concentration phosphate buffer (e.g., 2, 5, 10, 15,20, or 25 mM) and then a high concentration phosphate buffer (e.g., 30;50; 75; 100; 250; 500; 750; 1,000; 1,500; or 2,000 mM).

The pH of the phosphate containing buffer and the amount of thephosphate containing buffer passed through the resin can vary, but willgenerally be selected as any pH that will prevent or reduce thedeterioration of the resin that occurs prior to apatite use (e.g., priorto purification). Exemplary pH values suitable for apatite pretreatmentwith a phosphate containing buffer include any pH that is at least about5, at least about 5.5, at least about 6, at least about 6.5, at leastabout 7, at least about 7.5, at least about 8, or at least about 8.5, orhigher. In some cases, the pH of the phosphate containing buffer is 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or higher.

The volume of the solution needed to achieve the pretreatment can varywith the phosphate ion concentration, but in most cases best resultswill be achieved with from about 1.0 to about 10.0 resin volumes ofsolution, and in many cases with about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or about 3resin volumes. The volume can be up to about 6 resin volumes, including2, 3, 4, or 5 resin volumes. In some cases, a high phosphateconcentration at a volume that is less than a resin volume (e.g., lessthan about 0.9, 0.7, 0.5 volumes) can be utilized.

In some embodiments, the apatite solid surface is in a column, e.g., achromatography column, and the phosphate containing buffer can beapplied to the apatite solid surface at a flow rate. The flow rate canvary, but will generally be selected as any rate that will prevent orreduce deterioration of the resin that occurs prior to apatite use(e.g., prior to purification). Suitable flow rates include rates thatare typically used during equilibration or loading of apatite. Anexemplary flow rate is 400 cm/hr. In some cases, the flow rate issubstantially lower than 400 cm/hr (e.g., 300, 200, 100, or 50 cm/hr, orless). The use of a low flow rate can allow a greater contact timebetween the apatite solid surface and the phosphate containing buffer. Alow flow rate can be particularly preferred when the concentration ofphosphate, or the volume of the phosphate containing buffer, is low. Alow flow rate can also be preferred when the phosphate containing bufferis viscous. Alternatively, the flow rate can be higher than 400 cm/hr.In some cases, the formation of a loosely bound layer of phosphate israpid and a high flow rate can advantageously reduce the time requiredfor apatite pretreatment. In some cases, the formation of a looselybound layer of phosphate applies to a column having a diameter of 5centimeters or less.

In some embodiments, the apatite solid surface is contacted with thephosphate containing buffer in a batch format. In a batch format, thephosphate containing buffer can be applied by pouring the phosphatecontaining buffer onto the apatite solid surface, or pouring of theapatite solid surface into the phosphate containing buffer. The contacttime can vary, but will generally be selected as any time that willprevent or reduce deterioration of the resin that occurs prior toapatite use (e.g., prior to purification).

In some embodiments, the apatite solid surface is then washed or rinsed.In other embodiments, the apatite solid surface is not washed or rinsedafter pretreatment with a phosphate containing buffer. One of skill inthe art can readily select a suitable wash buffer. In some cases, theresin is treated with a wash solution to remove any excess phosphateions. Generally, the wash buffer is at a pH, composition, andconcentration that does not substantially leach components of theapatite surface, release accumulated hydronium ions, or generateundesirable precipitate. For example, the wash buffer can be compatible,and thus not precipitate when mixed, with the preceding and subsequentbuffer. As another example, the wash buffer can be selected that doesnot leach any loosely bound phosphate layer formed during the contactingof the apatite solid surface with the phosphate buffered solution.Suitable washing buffers can include buffer compositions typically usedfor equilibration of apatite. In some cases, the apatite solid surfaceis washed with a low molarity phosphate buffer (e.g., phosphate at aconcentration of less than about 100 mM, 50 mM, 25 mM, 20 mM, 15 mM, 10mM, or 5 mM). The pH of the wash buffer can be at least about 5, atleast about 5.5, at least about 6, or at least about 6.5, 7, or 8. Insome cases, a water wash is applied, and the amounts can vary widely. Atypical water wash will be at least about 0.2 resin volumes, and in mostcases from about 0.2 to about 1.5 or from about 0.2 to about 2 resinvolumes.

B. Hydroxide

The hydroxide ion treatment is applied as the last step of the apatitesolid surface pretreatment (e.g., the hydroxide ion is applied to theapatite solid surface after the phosphate buffered solution). Anysoluble form of hydroxide ion can be used, preferably water-soluble. Insome cases, alkali metal hydroxides, such as sodium or potassiumhydroxide, are particularly convenient. As in the case of the phosphateion, the concentration and quantity of hydroxide ion solution can vary.Without wishing to be bound by theory, it is believed that the hydroxideinteracts with the apatite solid surface, or loosely bound (e.g.,non-covalently bound) calcium, phosphate, or calcium and phosphatelayer(s), to convert the loosely bound (e.g., non-covalently bound)minerals into apatite, thus providing a pretreated surface. An amount,volume, concentration, etc. of hydroxide that will prevent or reduce thedeterioration of the resin that occurs prior to a purification procedurecan be an amount that allows for sufficient conversion of loosely boundcalcium, phosphate, or calcium phosphate to apatite. Accordingly, insome embodiments, the hydroxide ion solution does not contain calcium orphosphate.

The hydroxide ion concentration can be from about 0.005 or 0.01 M toabout 5 M; about 0.1 M to about 4.0 M, and in many cases from about 0.3M to about 3.0 M, including 0.2 M, 0.5 M, 0.75 M, 1.0 M, 1.25 M, 1.5 M,2.0 M, or 2.5 M. Suitable volumes of hydroxide ion containing treatmentsolution range from about 1.0 to about 20.0 resin volumes, and in manycases from about 1.5 to about 10.0 resin volumes, including 2, 3, 4,4.5, 5, 6, 7, 8, or 9 volumes. In some cases, a high hydroxideconcentration at a volume that is less than a resin volume (e.g., lessthan about 0.9, 0.7, 0.5 volumes) can be utilized.

Following hydroxide treatment, the resin can be washed or equilibratedwith a suitable buffer. In some cases, the resin is equilibrated, orwashed and then equilibrated, with a loading buffer. For example, theresin can be equilibrated with 10 mM phosphate buffer, pH 6.5 toequilibrate the column for protein purification. In some cases, theresin is equilibrated, or washed and then equilibrated, with a storagebuffer. For example, the resin can be equilibrated with 0.1 M NaOH, 10mM phosphate buffer, pH 6.5 and then stored.

III. Regeneration Methods

In some embodiments, a regeneration method is applied to the pretreatedapatite solid surface after each use in a chromatographic procedure. Insome embodiments, the regeneration method is applied intermittently tothe pretreated apatite solid surface after a chromatographic procedure,i.e., the regeneration method is applied to the pretreated apatite solidsurface after two or more purification procedures. In some embodiments,the regeneration method is applied to the pretreated apatite solidsurface after the first purification procedure and then after two ormore purification procedures.

Protein purification with an apatite resin can generally be performed intwo ways: (i) flow through purification; and (ii) bind and elutepurification. For flow through purification, traditionally, one (a)equilibrates the column in a suitable buffer; (b) adds a sample to acolumn under conditions in which impurities bind to the column and thetarget molecule flows through and is collected, (c) cleans, or strips,the column to remove adsorbed biological compounds with acleaning/stripping solution (e.g., a high molarity phosphate solution),and (d) regenerates, or sanitizes, the column with a strong alkalinehydroxide solution so that the column can be re-used. In some cases, thestrong alkaline hydroxide solution is replaced with a low molarity rinsefor long term storage or re-equilibration.

For bind and elute purification, traditionally, one (a) equilibrates thecolumn in a suitable buffer; (b) adds a sample to a column underconditions in which the target molecule binds to the column, (c) elutesthe target molecule (e.g., with a high molarity phosphate and/oralkaline halide solution), (d) cleans, or strips, the column to removeadsorbed biological compounds with a cleaning solution (e.g., a highmolarity phosphate solution), and (e) regenerates, or sanitizes, thecolumn with a strong alkaline hydroxide solution so that the column canbe re-used. In some cases, the strong alkaline hydroxide solution isreplaced with a low molarity rinse for long term storage orre-equilibration.

These traditional apatite purification methods can suffer from poorreproducibility and/or premature apatite deterioration. In some cases,this deterioration is due to the accumulation of hydronium ions (H3O+)on the apatite surface during exposure to equilibration, loading, orchromatography buffers. Hydronium ion accumulation can occur duringexposure to alkali metal salts at a pH of 8.0 or below. Hydronium ionaccumulation can also occur during exposure to phosphate buffers at a pHof less than about 6.5. Other buffer compositions can also causehydronium ion accumulation. These hydronium ions are then desorbed uponexposure to a subsequent buffer, such as an elution buffer (e.g., duringbind and elute purification) or a cleaning/stripping buffer (e.g., afterflow through purification). This desorption causes the resin todeteriorate over time, resulting in a loss of resin mass and/or adecline in the particle strength of the resin.

In some embodiments, a sample is contacted with a pretreated apatitesolid surface that is equilibrated with a suitable buffer (e.g., anequilibrated apatite solid surface). The target molecule is thencollected (e.g., during flow through purification, or after elution),and the apatite is regenerated by contacting the apatite solid surfacewith a buffered calcium solution, followed by a phosphate bufferedsolution, followed by an alkaline hydroxide. In some cases, the apatitesolid surface is used multiple times (i.e., at least two times) fortarget analyte purification prior to application of one or moreregeneration steps described herein.

In some embodiments, the apatite solid surface is washed or rinsed priorto regenerating. In other embodiments, the apatite solid surface is notwashed or rinsed prior to regenerating. In some cases, the resin istreated with a wash solution to remove any excess calcium, phosphate, orhydroxide ions. One of skill in the art can readily select a suitablewash buffer. Generally, the wash buffer can be at a pH, composition, andconcentration that does not substantially leach components of theapatite surface, release accumulated hydronium ions, or generateundesirable precipitate. For example, the wash buffer can be compatible,and thus not precipitate when mixed, with the preceding and subsequentbuffer. Suitable washing buffers can include buffer compositionstypically used for equilibration, loading, or flow through of apatite.In some cases, the apatite solid surface is washed with a low molarityphosphate buffer (e.g., phosphate at a concentration of less than about100 mM, 50 mM, 25 mM, 20 mM, 15 mM, 10 mM, or 5 mM). The pH of the washbuffer can be at least about 5, at least about 5.5, at least about 6, orat least about 6.5, 7, or 8. An exemplary wash buffer pH is 5.5, 6, or6.5. In some cases, a water wash is applied, and the amounts can varywidely. A typical water wash will be at least about 0.2 resin volumes,and in most cases from about 0.2 to about 1.5 or from about 0.2 to about2 resin volumes.

The apatite solid surface can then be regenerated. In some cases, theapatite solid surface can be regenerated, e.g., after elution, afterflow through, after neutralization, after cleaning/stripping, afterrinsing, or after storage. In some cases, the apatite solid surface canbe regenerated after a wash, e.g., after application of a wash buffer toremove a flow through, elution, neutralization, rinsing, storage, orcleaning/stripping buffer.

A. Buffered Calcium Solution

The regeneration begins with contacting the apatite solid surface with abuffered calcium solution. Although, regeneration of the apatite solidsurface has been attempted using an unbuffered calcium solution, thepresent inventors have found that the use of a buffered calcium solutionappears to significantly and surprisingly enhance the degree ofregeneration obtained. The calcium ion concentration of the bufferedcalcium solution and the amount of the buffered calcium solution passedthrough the resin can vary, but will generally be selected as any amountthat will reduce, eliminate, or reverse the deterioration of the resinthat occurs during apatite use (e.g., during purification, duringelution, or during cleaning/stripping).

Without wishing to be bound by theory, it is believed that the bufferedcalcium solution interacts with the apatite solid surface to generate aloosely bound (e.g., non-covalent) calcium layer on the apatite solidsurface. In some cases, this calcium layer replaces some or all (or morethan all) of the calcium lost during previous purification steps. Thus,an amount, volume, concentration, etc. of calcium ion, or any othercomponent or aspect of the buffered calcium solution that will reduce,eliminate, or reverse the deterioration of the resin that occurs duringapatite use, can be an amount that allows for sufficient formation of aloosely bound calcium layer.

The calcium ion concentration is generally selected to be below thesolubility limit of the calcium at the pH and temperature of thebuffered calcium solution. Moreover, the concentration can vary based onthe presence, absence, or concentration of other components in thebuffered calcium solution, such as the selected buffering agent, orbased on the selected composition of any preceding buffer. In certainembodiments of the concepts herein, best results will be achieved with acalcium ion concentration of from about 5 mM, 5.1 mM, 5.2 mM, 5.3 mM,5.4 mM, 5.5 mM, 5.6 mM, 5.7 mM, 5.8 mM, 5.9 mM, 6 mM, 6.5 mM, 7 mM, 8mM, 9 mM, 10 mM, 10.1 mM, 10.2 mM, 10.3 mM, 10.5 mM or 11 mM to about 15mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 75 mM, 100 mM or 250 mM. Incertain embodiments, the calcium ion concentration in the buffer calciumsolution is from about 5 mM to about 10 mM, from about 5 mM to about 25mM, from about 20 mM to about 100 mM, or from about 25 mM to about 50-75mM, including 5 mM, 5.1 mM, 5.2 mM, 5.3 mM, 5.4 mM, 5.5 mM, 5.6 mM, 5.7mM, 5.8 mM, 5.9 mM, 6 mM, 6.5 mM, 7 mM, 8 mM, 9 mM, 10 mM, 10.1 mM, 10.2mM, 10.3 mM, 10.5 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 20 mM, 25 mM,30 mM, 40 mM, 60 mM, 70 mM, 80 mM, 90 mM, 110 mM, 150 mM, 200 mM, 300mM, or higher.

The volume of the solution needed to achieve the restoration can varywith the calcium ion concentration, but in most cases best results willbe achieved with from about 1.0 to about 10.0 resin volumes of solution,and in many cases with about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, or about 2 resin volumes. In some cases, the volume can be up toabout 6 resin volumes, including 2, 3, 4, or 5 resin volumes. In somecases, the volume is less than 3 column volumes. In some cases, a highcalcium ion concentration at a volume that is less than a resin volume(e.g., less than about 0.9, 0.7, 0.5 volumes) can be utilized.

A wide variety of buffers are suitable for the buffered calcium solutionfor apatite regeneration. In some embodiments, a buffer for the bufferedcalcium solution that does not appreciably form metal complexes insolution (e.g., does not form a complex with calcium at the pH of thebuffer solution) can comprise the buffer component of the bufferedcalcium solution. In some embodiments, a buffer that does not containprimary or secondary (i.e., R₂-N, wherein R is not H) amine can comprisethe buffer component of the buffered calcium solution. In someembodiments, a zwitterionic buffer is preferred. In some embodiments, abuffer (e.g., a zwitterionic buffer) that contains a sulfonic acidmoiety is preferred. In some embodiments, a buffer (e.g., a zwitterionicbuffer) that contains a sulfonic acid and a tertiary amine (i.e., R₃-N,wherein R is not H) is preferred. Exemplary zwitterionic bufferssuitable for use as a buffering agent in the buffered calcium solutioninclude, but are not limited to, one or more of the following: MES,PIPES, ACES, MOPSO, MOPS, BES, TES, HEPES, DIPSO, TAPS, TAPSO, POPSO, orHEPPSO, EPPS, CAPS, CAPSO, CHES, MOBS, TABS, or AMPSO. In someembodiments, the buffer of the buffered calcium solution contains aprimary, secondary, or a tertiary amine. In some embodiments, the bufferof the buffered calcium solution contains a primary, secondary or atertiary amine and a one or more carboxylate or hydroxymethyl groups. Insome embodiments, the buffer of the buffered calcium solution istricine, bicine, or Tris. In some embodiments, the buffer of thebuffered calcium solution isbis(2-hydroxyethyl)-amino-tris(hydroxymethyl)-methane), or 1,3-bis(tris(hydroxymethyl) methylamino) propane.

The buffer concentration in the buffered calcium solution can vary, butwill generally be selected as a concentration that is at least as highas the calcium ion concentration of the solution. Moreover, theconcentration can vary based on the selected buffering agent, or theselected composition of any preceding buffer. Thus, the ratio of thebuffer concentration to the calcium ion concentration is generally atleast about 1, e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, or higher. Generally, the bufferconcentration is also selected such that it is below the solubilitylimit of the buffering agent. In some cases, preferred buffering agentsinclude those that have a high solubility limit.

The pH of the buffered calcium solution can vary, but will generally beselected as any amount that will reduce, eliminate, or reversedeterioration of the resin that occurs during apatite use (e.g., duringpurification, during elution, or during cleaning/stripping). Moreover,the pH can vary based on the selected apatite solid surface, theselected buffering agent, the selected concentration of one or morecomponents, or the selected composition of any preceding buffer.Typically, the pH is at least about 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 6,6.2, 6.5, 7, 7.5, or 8. In some embodiments, the pH is, or is at leastabout 5.5, 6, 6.5, 7, 7.5, or 8. In some embodiments, the pH is 5.5, 6,6.5, 7, 7.5, or 8. In some embodiments, the pH is 5.1, 5.2, 5.3, or 5.4.In some cases, the pH is 5.3. In some cases, the pH is 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, or 7.5. In some cases, the pH is 7.0. In some cases, the pH is5.6. In some cases, the pH is 6.2. In some cases, the pH is 5.4.

In some embodiments, the buffer of the buffered calcium solution is aphosphate buffer. In such cases, the calcium and phosphateconcentrations and the pH of the solution can be selected to provideregeneration while avoiding precipitant formation, or avoiding asupersaturated solution. For example, the pH of the phosphate bufferedcalcium solution can be selected to be sufficiently low (e.g., a pH ofabout, or less than about, 6.5, 6.4, 6.3, 6.2, 6.1, 6, 5.9, 5.8, 5.7,5.6, 5.5, 5.4, 5.3, 5.2, 5.1, or 5). In some cases, the pH is 5.1, 5.2,5.3, 5.4, or 5.5. In some cases, the pH is 5.3. As another example, thecalcium concentration of the phosphate buffered calcium solution can beabout, or less than about, 50 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15mM, 10 mM, 7 mM, 6 mM, 5.9 mM, 5.8 mM, 5.7 mM, 5.6 mM, 5.5 mM, 5.4 mM,5.3 mM, 5.2 mM, 5.1 mM, or 5 mM. In some cases, the calciumconcentration of the phosphate buffered solution is, or is about, 15 mM,14 mM, 13 mM, 12 mM, 11 mM, 10.5 mM, 10.4 mM, 10.3 mM, 10.2 mM, 10.1 mM,10 mM, or 9.5 mM. In some cases, the calcium concentration is 10 or 10.2mM. In some cases, the calcium concentration is 10 mM. As anotherexample, the phosphate concentration of the phosphate buffered calciumsolution can be about, or less than about, 50 mM, 40 mM, 35 mM, 30 mM,29 mM, 28 mM, 27 mM, 26 mM, 25 mM, 24 mM, 23 mM, 21 mM, 20 mM, 18 mM, 17mM, 16 mM, or 15 mM. In some cases, the use of a phosphate bufferedcalcium solution provides regeneration with or without a preceeding orsubsequent high molarity (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, or 1 M) phosphate buffer step.

In some embodiments, the apatite solid surface is in a column, e.g., achromatography column, and the buffered calcium solution can be appliedto the apatite solid surface at a flow rate. The flow rate can vary, butwill generally be selected as any rate that will reduce, eliminate, orreverse deterioration of the resin that occurs during apatite use (e.g.,during purification, during elution, or during cleaning/stripping).Suitable flow rates, include rates that are typically used duringequilibration, loading, elution, cleaning/stripping, sanitation, orrinsing of apatite. An exemplary flow rate is 400 cm/hr. In some cases,the flow rate is substantially lower than 400 cm/hr (e.g., 300, 200,100, or 50 cm/hr, or less). The use of a low flow rate can allow agreater contact time between the apatite solid surface and the bufferedcalcium solution. A low flow rate can be particularly preferred when theconcentration of calcium or buffering agent, or the volume of thebuffered calcium solution, is low. A low flow rate can also be preferredwhen the buffered calcium solution, or the preceding solution, isviscous or the column is fouled with adsorbed biological compounds.Alternatively, the flow rate can be higher than 400 cm/hr. In somecases, the formation of a loosely bound layer of calcium is rapid and ahigh flow rate can advantageously reduce the time required for apatiteregeneration.

In some embodiments, the apatite solid surface is contacted with thebuffered calcium solution in a batch format. In a batch format, thebuffered calcium solution can be applied by pouring the buffered calciumsolution onto the apatite solid surface, or pouring a slurry of theapatite solid surface into the buffered calcium solution. The contacttime can vary, but will generally be selected as any time that willreduce, eliminate, or reverse deterioration of the resin that occursduring apatite use (e.g., during purification, during elution, or duringcleaning/stripping).

In some embodiments, the apatite solid surface is then washed or rinsed.One of skill in the art can readily select a suitable wash buffer. Insome cases, the resin is treated with a wash solution between theindividual regeneration treatments to remove any excess calcium,phosphate, or hydroxide ions. Generally, the wash buffer can be at a pH,composition, and concentration that does not substantially leachcomponents of the apatite surface, release accumulated hydronium ions,or generate undesirable precipitate. For example, the wash buffer can becompatible, and thus not precipitate when mixed, with the preceding andsubsequent buffer. As another example, the wash buffer can be selectedthat does not leach any loosely bound calcium layer formed during thecontacting of the apatite solid surface with the buffered calciumsolution. Suitable washing buffers can include buffer compositionstypically used for equilibration, loading, or flow through of apatite.In some cases, the apatite solid surface is washed with a low molarityphosphate buffer (e.g., phosphate at a concentration of less than about100 mM, 50 mM, 25 mM, 20 mM, 15 mM, 10 mM, or 5 mM). The pH of the washbuffer can be at least about 5, at least about 5.5, at least about 6, orat least about 6.5, 7, or 8. In some cases, a water wash is applied, andthe amounts can vary widely. A typical water wash will be at least about0.2 resin volumes, and in most cases from about 0.2 to about 1.5 or fromabout 0.2 to about 2 resin volumes.

B. Phosphate Buffered Solution

The apatite solid surface can then be contacted with a phosphatecontaining buffer after the apatite has been contacted with a bufferedcalcium solution. Alternatively, the phosphate containing buffer can becontacted with apatite before the apatite has been contacted with abuffered calcium solution. In some cases, an intervening wash step isapplied between the buffered calcium solution and the phosphatecontaining buffer. The phosphate concentration of the phosphatecontaining buffer and the amount of the phosphate containing bufferpassed through the resin can vary, but will generally be selected as anyamount that will reduce, eliminate, or reverse the deterioration of theresin that occurs during apatite use (e.g., during purification, duringelution, or during cleaning/stripping) and can be an amount that allowsfor sufficient formation of a loosely bound phosphate layer.

The phosphate concentration of the phosphate containing buffer isgenerally selected to be below the solubility limit of the phosphate atthe pH and temperature of the buffer. Moreover, the concentration canvary based on presence or absence of other components of the buffer, orthe selected composition of any preceding buffer. In certain embodimentsof the concepts herein, best results will be achieved with a phosphateconcentration of from about 10 mM to about 1, 1.5, or 2 M; from about 20mM to about 1.5 M; or from about 25 mM to about 1 M; from about 50 mM toabout 1 M; including at least about, or about, 10 mM, 15 mM, 20 mM, 25mM, 30 mM, 40 mM, 50 mM, 60 ppm, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM,150 mM, 200 mM, 300 mM, 500 mM, 750 mM, 1 M, or higher. In some cases,the phosphate concentration is 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM,50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 150 mM, 200 mM, 300mM, 500 mM, 750 mM, 1 M, or higher. In some cases, the phosphateconcentration is from, or from about, 0.1 or 2 M to, or to about, 0.4 M,0.5 M, or 1 M. In some cases, the column is contacted with a lowconcentration phosphate buffer (e.g., 2, 5, 10, 15, 20, or 25 mM) andthen a high concentration phosphate buffer (e.g., 30; 50; 75; 100; 250;500; 750; 1,000; 1,500; or 2,000 mM). In some cases, the use of a lowconcentration phosphate buffer followed by a high concentrationphosphate buffer can avoid potential incompatibility (e.g.,precipitation) between the buffered calcium solution and the highconcentration phosphate buffer.

The pH of the phosphate containing buffer and the amount of thephosphate containing buffer passed through the resin can vary, but willgenerally be selected as any pH that will reduce, eliminate, or reversethe deterioration of the resin that occurs during apatite use (e.g.,during purification, during elution, or during cleaning/stripping).Exemplary pH values suitable for apatite regeneration with a phosphatecontaining buffer include any pH that is at least about 5, at leastabout 5.5, at least about 6, at least about 6.5, at least about 7, atleast about 7.5, at least about 8, or at least about 8.5, or higher. Insome cases, the pH of the phosphate containing buffer is 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10, or higher.

The volume of the solution needed to achieve the restoration can varywith the phosphate ion concentration, but in most cases best resultswill be achieved with from about 1.0 to about 10.0 resin volumes ofsolution, and in many cases with about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, or about 2 resin volumes. The volume can be up to about 6resin volumes, including 2, 3, 4, or 5 resin volumes. In some cases, ahigh phosphate concentration at a volume that is less than a resinvolume (e.g., less than about 0.9, 0.7, 0.5 volumes) can be utilized.

In some embodiments, the apatite solid surface is in a column, e.g., achromatography column, and the phosphate containing buffer can beapplied to the apatite solid surface at a flow rate. The flow rate canvary, but will generally be selected as any rate that will reduce,eliminate, or reverse deterioration of the resin that occurs duringapatite use (e.g., during purification, during elution, or duringcleaning/stripping). Suitable flow rates, include rates that aretypically used during equilibration, loading, elution,cleaning/stripping, sanitation, or rinsing of apatite. An exemplary flowrate is 400 cm/hr. In some cases, the flow rate is substantially lowerthan 400 cm/hr (e.g., 300, 200, 100, or 50 cm/hr, or less). The use of alow flow rate can allow a greater contact time between the apatite solidsurface and the phosphate containing buffer. A low flow rate can beparticularly preferred when the concentration of phosphate, or thevolume of the phosphate containing buffer, is low. A low flow rate canalso be preferred when the phosphate containing buffer, or the precedingsolution, is viscous or the column is fouled with adsorbed biologicalcompounds. Alternatively, the flow rate can be higher than 400 cm/hr. Insome cases, the formation of a loosely bound layer of phosphate is rapidand a high flow rate can advantageously reduce the time required forapatite regeneration.

In some embodiments, the apatite solid surface is contacted with thephosphate containing buffer in a batch format. In a batch format, thephosphate containing buffer can be applied by pouring the phosphatecontaining buffer onto the apatite solid surface, or pouring a slurry ofthe apatite solid surface into the phosphate containing buffer. Thecontact time can vary, but will generally be selected as any time thatwill reduce, eliminate, or reverse deterioration of the resin thatoccurs during apatite use (e.g., during purification, during elution, orduring cleaning/stripping).

In some embodiments, the apatite solid surface is then washed or rinsed.In other embodiments, the apatite solid surface is not washed or rinsedafter regeneration treatment with a phosphate containing buffer. One ofskill in the art can readily select a suitable wash buffer. In somecases, the resin is treated with a wash solution to remove any excesscalcium, phosphate, or hydroxide ions. Generally, the wash buffer is ata pH, composition, and concentration that does not substantially leachcomponents of the apatite surface, release accumulated hydronium ions,or generate undesirable precipitate. For example, the wash buffer can becompatible, and thus not precipitate when mixed, with the preceding andsubsequent buffer. As another example, the wash buffer can be selectedthat does not leach any loosely bound calcium layer formed during thecontacting of the apatite solid surface with the buffered calciumsolution. Suitable washing buffers can include buffer compositionstypically used for equilibration, loading, or flow through of apatite.In some cases, the apatite solid surface is washed with a low molarityphosphate buffer (e.g., phosphate at a concentration of less than about100 mM, 50 mM, 25 mM, 20 mM, 15 mM, 10 mM, or 5 mM). The pH of the washbuffer can be at least about 5, at least about 5.5, at least about 6, orat least about 6.5, 7, or 8. In some cases, a water wash is applied, andthe amounts can vary widely. A typical water wash will be at least about0.2 resin volumes, and in most cases from about 0.2 to about 1.5 or fromabout 0.2 to about 2 resin volumes.

A degree of resin regeneration can be achieved with either the bufferedcalcium solution treatment preceding the phosphate containing buffertreatment, or with the phosphate containing buffer treatment precedingthe buffered calcium solution treatment. In some embodiments, a greaterdegree of regeneration can be achieved by applying the buffered calciumsolution treatment first, followed by the phosphate containing buffertreatment. In some embodiments, a preferred degree of regeneration canbe achieved by performing one or more steps of buffered calcium solutiontreatment subsequent to, or followed by, one or more steps of phosphatecontaining buffer treatment. In some cases, one or more of multiplesteps of buffered calcium solution treatment or phosphate containingbuffer treatment are preceded by or followed by a wash.

In some embodiments, the buffered calcium solution treatment and/or thephosphate containing buffer treatment is applied after elution or flowthrough of a target analyte. For example, an apatite surface can beequilibrated, contacted with a target analyte, the target analyte can beeluted or collected in the flow through, and then the regenerationprotocol can be applied. As described herein, exemplary regenerationprotocols can include, but are not limited to, those in which a bufferedcalcium solution is contacted with the apatite solid surface and then aphosphate buffer is contacted with the apatite solid surface. Exemplaryregeneration protocols can further include, but are not limited to,those in which a phosphate regeneration buffer is contacted with theapatite solid surface and then a buffered calcium regeneration solutionis contacted with the apatite solid surface. An alkaline hydroxidetreatment can be applied after the apatite is contacted with thebuffered calcium and phosphate regeneration solutions.

C. Hydroxide

The hydroxide ion treatment is applied as the last treatment step of theapatite solid surface regeneration. Any soluble form of hydroxide ioncan be used, preferably water-soluble. In some cases, alkali metalhydroxides, such as sodium or potassium hydroxide, are particularlyconvenient. As in the cases of the calcium ion and the phosphate ion,the concentration and quantity of hydroxide ion solution can vary. Anamount, volume, concentration, etc. of hydroxide that will reduce,eliminate, or reverse the deterioration of the resin that occurs duringapatite use can be an amount that allows for sufficient conversion ofloosely bound calcium, phosphate, or calcium phosphate to apatite. Thehydroxide ion can also clean the resin of residual proteins andcontaminants and can also serve as a sanitation or storage solution.

The hydroxide ion concentration can be from about 0.005 or 0.01 M toabout 5 M; about 0.1 M to about 4.0 M, and in many cases from about 0.3M to about 3.0 M, including 0.2 M, 0.5 M, 0.75 M, 1.0 M, 1.25 M, 1.5 M,2.0 M, or 2.5 M. Suitable volumes of hydroxide ion containing treatmentsolution range from about 1.0 to about 20.0 resin volumes, and in manycases from about 1.5 to about 10.0 resin volumes, including 2, 3, 4,4.5, 5, 6, 7, 8, or 9 volumes. In some cases, a high hydroxideconcentration at a volume that is less than a resin volume (e.g., lessthan about 0.9, 0.7, 0.5 volumes) can be utilized.

Following hydroxide treatment, the resin can be washed or equilibratedwith a suitable buffer. In some cases, the resin is equilibrated, orwashed and then equilibrated, with a loading buffer. For example, theresin can be equilibrated with 10 mM phosphate buffer, pH 6.5 toequilibrate the column for protein purification. In some cases, theresin is equilibrated, or washed and then equilibrated, with a storagebuffer. For example, the resin can be equilibrated with 0.1 M NaOH, 10mM phosphate buffer and then stored.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially the same or similar results.

Example 1

This example illustrates the deterioration (i.e., leaching of calcium)of a hydroxyapatite resin prior to first use in a purificationprocedure.

Four experiments were conducted to determine the extent of calciumleaching of four contact solutions: (1) 0.4 M NaPO4 at pH 8.8, (2) 1 MNaCl, (3) PBS at pH 7.4 (12 mM phosphate, 0.15 M NaCl, pH 7.4), and (4)0.4 M NaPO4 at pH 6.6. The experiment was performed with columnsmeasuring 20 cm in length and 3.2 cm in internal diameter, with aninternal volume of 159 mL. Each column was dry packed with 100 g ofceramic hydroxyapatite Type I powder, then hydrated by pumping 240 mL ofthe contact solution into the column inlet and stopping just prior tothe effluent exiting the column. The column exit was connected toconductivity and pH flow monitors, then to a fraction collector. Thecolumn was eluted at 140 cm/hour while collecting 6×0.25 column volumefractions of effluent (240 mL). The amount of hydrate (160 mL) andcollected contact solution (i.e., total solution volume of 400 mL or 2.5column volumes) is equivalent to the amount of solution recommended fora mixture of ceramic hydroxyapatite/contact solution in preparation forhydrated packing of process columns. Table I below lists the pH ofeffluent fractions, the calcium concentration for each fraction, and theestimated calcium leaching in a typical slurry mixture used for processchromatography column packing. The calcium concentration was determinedwith a colorimetric assay (BioAssay Systems kit DICA-500 or theBiovision kit K380-250).

TABLE I Calcium Effluent Analysis and Estimated Calcium Level in SlurryMixtures Solution Calcium Volume concentration (400 mL) Contact SolutionID pH ppm ppm 0.4M NaPO4, pH 8.8 6141-083 A1 5.95 56 6 A2 7.41 0 A3 7.930 A4 8.13 0 A5 8.31 0 A6 8.43 0 1M NaCl 6141-083 B1 5.13 1576 339 B25.29 739 B3 5.45 402 B4 5.54 263 B5 5.60 228 B6 5.65 180 PBS, pH 7.46141-083 C1 5.90 425 131 C2 5.76 356 C3 5.76 208 C4 5.76 158 C5 5.76 114C6 5.76 43 0.4M NaPO4, pH 6.6 6141-083 D1 5.55 105 14 D2 6.53 8 D3 6.595 D4 6.62 5 D5 6.57 5 D6 6.58 5

The data in Table I show that PBS, pH 7.4 and 1 M NaCl contact solutionsleach large amounts of calcium, whereas contact solutions having 0.4 Mphosphate at either pH 6.6 or 8.8 do not leach large amounts of calcium.The results confirm that phosphate contact solution minimizes calciumleaching while hydrating the hydroxyapatite powder.

Example 2

This example illustrates the result of incorporating an intermittent orcontinuous in situ regeneration (ISR) protocol after a pretreatmentprotocol. The apatite resin is exposed to a series of cycles thatsimulate conditions encountered in protein separation, but withoutloading and eluting protein.

All columns used in the following eight experiments underwent apretreatment protocol (i.e., all columns were dry packed and thenequilibrated with 0.4 M sodium phosphate buffer, pH 8.8 followed byeither 0.5 M or 1 M sodium hydroxide) prior to the experiments describedbelow. The description and conditions for the eight experiments arelisted in Tables II through IX below. A series of consecutive cycleswere performed for each experiment, each cycle consisting of the stepsindicated in each table. Experiments outlined in Tables II-VI used 30cycles each. The experiment outlined in Table VII used 10 cycles. Theexperiments outlined in Tables VIII and IX used 24 cycles and 5 cycles,respectively.

The experiments outlined in Tables II-III and V-VII were each performedon a column measuring 30 cm in length and 1.6 cm internal diameter withan internal volume of 60.32 mL. The packing was ceramic hydroxyapatiteType I in 40-micron particles weighing 38 grams and the resulting mobilephase flow rate through the column being 180 cm/hour. The experimentoutlined in Tables IV, VIII and IX were performed on a column having alength of 20 cm, a 1.6 cm internal diameter, an internal volume of 40.21mL and with a mobile phase flow rate of 350 cm/hour. The packing wasceramic hydroxyapatite Type I in 40-micron particles weighing 25.33grams. Mobile phase entry was at the top of each of the columns.

Tables II-IV simulate control purification protocols without columnrestoration. In step 3 of the Table III control protocol, polysaccharideis loaded onto the column. The control protocol in Table IV simulates anacidic protein elution with a shallow phosphate gradient (see step 6).

In Tables IV, VIII and IX, dilute WS refers to an WS concentration of atleast about 10 mM, 15 mM, or 20 mM IVIES and less than about 25 mM or 30mM WS. Dilute Tris refers to a Tris concentration of at least about 2mM, 3 mM, 4 mM, 5 mM, 10 mM, or 15 mM Tris and less than about 20 mM, or25 mM Tris.

TABLE II Control Treatment Protocol Using 30 Cycles Amount Column VolumeStep Description Mobile Phase Volumes in mL 1Pre-Equilibration/Regeneration 0.5M NaPO4, pH 7.0 2.5 150.8 2Equilibration 25 mM NaPO4, 250 mM NaCl, pH 7.0 2.5 150.8 3 Product load25 mM NaPO4, 250 mM NaCl, pH 7.0 3.0 181.0 4 Product recovery flush 25mM NaPO4, 250 mM NaCl, pH 7.0 2.5 150.8 5 Pre-Equilibration/Regeneration0.5M NaPO4, pH 7.0 5.0 301.6 6 Sanitization 0.5M NaOH 4.5 271.4

TABLE III Control Treatment Protocol Using 30 Cycles and Simulating aSaccharide Load Amount Column Volume Step Description Mobile PhaseVolumes in mL 1 Pre-Equilibration/Regeneration 0.5M NaPO4, pH 7.0 2.5150.8 2 Equilibration 25 mM NaPO4, 250 mM NaCl, pH 7.0 2.5 150.8 3Product load 25 mM NaPO4, 250 mM NaCl, pH 3.0 181.0 7.0, 2% DEX T500 4Product recovery flush 25 mM NaPO4, 250 mM NaCl, pH 7.0 0.5 30.2 5Product recovery flush 25 mM NaPO4, 250 mM NaCl, pH 7.0 2.5 150.8 6Pre-Equilibration/Regeneration 0.5M NaPO4, pH 7.0 5.0 301.6 7Sanitization 0.5M NaOH 4.5 271.4

TABLE IV Control Treatment Protocol Using 30 Cycles and Simulating anAcidic Protein Elution Amount Column Volume Step Description MobilePhase Volumes in mL 1 Equilibration 1 50 mM NaPO4, 0.1M NaCl, pH 6.7 1.040.2 2 Equilibration 1 50 mM NaPO4, 0.1M NaCl, pH 6.7 4.0 160.8 3Equilibration 2 2 mM NaPO4, dilute MES, 0.1M 3.0 120.6 NaCl, pH 6.7 4Load 2 mM NaPO4, dilute MES, dilute Tris, 7.0 281.5 0.1M NaCl, pH 6.7 5Post-load wash 2 mM NaPO4, 0.1M NaCl, pH 6.5 3.0 120.6 6 Gradient 10%Equilibration 1, 90% 0.1M NaCl 10.0 402.1 --> 90% Equilibration 1, 10%0.1M NaCl 7 Strip with Equilibration 1 50 mM NaPO4, 0.1M NaCl, pH 6.72.0 80.4 8 Sanitization 1M NaOH 2.0 80.4

Tables V and VI are restoration protocols that use a low concentrationof buffer and calcium chloride in the buffered calcium step (i.e., step6 in each protocol).

TABLE V Column Restoration Protocol Amount Column Volume StepDescription Mobile Phase Volumes in mL 1 Pre-Equilibration/Regeneration0.5M NaPO4, pH 7.0 2.5 150.8 2 Equilibration 25 mM NaPO4, 250 mM NaCl,pH 7.0 2.5 150.8 3 Product load 25 mM NaPO4, 250 mM NaCl, pH 7.0 3.0181.0 4 Product recovery flush 25 mM NaPO4, 250 mM NaCl, pH 7.0 2.5150.8 5 Rinse Water 1.0 60.3 6 In-Situ Restoration 20 mM ACES, 5 mMCaCl2, pH 7.75 8.0 482.5 7 Rinse Water 1.0 60.3 8Pre-Equilibration/Regeneration 0.5M NaPO4, pH 7.0 5.0 301.6 9Sanitization 0.5M NaOH 4.5 271.4

TABLE VI Column Restoration Protocol Amount Column Volume StepDescription Mobile Phase Volumes in mL 1 Pre-Equilibration/Regeneration0.5M NaPO4, pH 7.0 2.5 150.8 2 Equilibration 25 mM NaPO4, 250 mM NaCl,pH 7.0 2.5 150.8 3 Product load 25 mM NaPO4, 250 mM NaCl, pH 7.0 3.0181.0 4 Product recovery flush 25 mM NaPO4, 250 mM NaCl, pH 7.0 2.5150.8 5 Rinse Water 1.0 60.3 6 In-Situ Restoration 20 mM MES, 5 mMCaCl2, pH 7.80 8.0 482.5 7 Rinse Water 1.0 60.3 8Pre-Equilibration/Regeneration 0.5M NaPO4, pH 7.0 5.0 301.6 9Sanitization 0.5M NaOH 4.5 271.4

Table VII is an intermittent restoration protocol that was applied afterpretreatment of the resin and after applying a control treatmentprotocol as outlined in Table II. The intermittent restoration protocolincludes a restoration protocol (i.e., steps R1-R7 in Table VII)followed by two control protocols (i.e., steps C1-C4 in Table VII). Thesteps listed in Table VII were repeated 10 times resulting in a total of10 restoration protocols and 20 control protocols.

TABLE VII Intermittent Column Restoration Protocol Amount Column VolumeStep Description Mobile Phase Volumes in mL R1Pre-Equilibration/Regeneration 0.5M NaPO4, pH 7.0 2.5 150.8 R2Equilibration-Load-Flush 25 mM NaPO4, 250 mM NaCl, pH 7.0 8.0 482.5 R3Rinse Water 1.0 60.3 R4 In-Situ Restoration 20 mM ACES, 5 mM CaCl2, pH7.75 8.0 482.5 R5 Rinse Water 1.0 60.3 R6 Pre-Equilibration/Regeneration0.5M NaPO4, pH 7.0 5.0 301.6 R7 Sanitization 0.5M NaOH 4.5 271.4 C1Pre-Equilibration/Regeneration 0.5 NaPO4, pH 7.0 2.5 150.8 C2Eq-Load-Flush 25 mM NaPO4, 250 mM NaCl, pH 7.0 8.0 482.5 C3Pre-Equilibration/Regeneration 0.5M NaPO4, pH 7.0 5.0 301.6 C4Sanitization 0.5M NaOH 4.5 271.4 C1 Pre-Equilibration/Regeneration 0.5MNaPO4, pH 7.0 2.5 150.8 C2 Equilibration/Load/Flush 25 mM NaPO4, 250 mMNaCl, pH 7.0 8.0 482.5 C3 Pre-Equilibration/Regeneration 0.5M NaPO4, pH7.0 5.0 301.6 C4 Sanitization 0.5M NaOH 4.5 271.4

Tables VIII and IX are column restoration protocols that include a highconcentration of buffer and calcium chloride in the buffered calciumsolution (see Step 9) than the restoration protocols in Tables V-VII(i.e., 50 mM CaCl2/100 mM MES/pH 7.0 versus 5 mM CaCl2/20 mM ACES orMES/pH 7.8). The protocol in Table VIII uses less column volumes andmore cycles of the stronger buffered calcium solution than the protocolin Table IX (i.e., 1.1 column volumes/24 cycles for the protocol inTable VIII versus 3.0 column volumes/5 cycles for the protocol in TableIX).

TABLE VIII Column Restoration Protocol Amount Column Volume in StepDescription Mobile Phase Volumes mL 1 Equilibration 1 50 mM NaPO4, 0.1MNaCl, pH 1.0 40.2 6.7 2 Equilibration 1 50 mM NaPO4, 0.1M NaCl, pH 4.0160.8 6.7 3 Equilibration 2 2 mM NaPO4, dilute MES, 0.1M 3.0 120.6 NaCl,pH 6.7 4 Load 2 mM NaPO4, dilute MES, 7.0 281.5 dilute Tris, 0.1M NaCl,pH 6.7 5 Post-load wash 2 mM NaPO4, 0.1M NaCl, pH 3.0 120.6 6.5 6Gradient 10% Equilibration 1, 90% 0.1M 10.0 402.1 NaCl --> 90%Equilibration 1, 10% 0.1M NaCl 7 Strip with Equilibration 1 50 mM NaPO4,0.1M NaCl, pH 2.0 80.4 6.7 8 Equilibration 2 2 mM NaPO4, dilute MES,0.1M 0.20 8.04 NaCl, pH 6.7 9 In-Situ Restoration 100 mM MES, 50 mMCaCl2, 1.1 44.2 pH 7.0 10 Equilibration 2 2 mM NaPO4, dilute MES, 0.1M0.20 8.04 NaCl, pH 6.7 11 Pre-Equilibration/Regeneration 400 mM NaPO4,pH 7.0 2.0 80.4

TABLE IX Column Restoration Protocol Amount Column Volume StepDescription Mobile Phase Volumes in mL 1 Equilibration 1 50 mM NaPO4,0.1M NaCl, pH 6.7 1.0 40.2 2 Equilibration 1 50 mM NaPO4, 0.1M NaCl, pH6.7 4.0 160.8 3 Equilibration 2 2 mM NaPO4, dilute MES, 0.1M 3.0 120.6NaCl, pH 6.7 4 Load 2 mM NaPO4, dilute MES, dilute Tris, 7.0 281.5 0.1MNaCl, pH 6.7 5 Post-load wash 2 mM NaPO4, 0.1M NaCl, pH 6.5 3.0 120.6 6Gradient Gradient 10-90% Equil-1 against 0.1M 10.0 402.1 NaCl 7 Stripwith Equilibration 1 50 mM NaPO4, 0.1M NaCl, pH 6.7 2.0 80.4 8Equilibration 2 2 mM NaPO4, dilute MES, 0.1M 0.20 8.04 NaCl, pH 6.7 9In-Situ Restoration 100 mM MES 50 mM CaCl2, pH 7.0 3.0 120.6 10Equilibration 2 2 mM NaPO4, dilute MES, 0.1M 0.20 8.04 NaCl, pH 6.7 11Pre-Equilibration/Regeneration 400 mM NaPO4, pH 7.0 2.0 80.4 12Sanitization 1M NaOH 2.0 80.4

The particle mass and particle strength was measured before the firstcycle and after the last cycle for each protocol described in TablesII-IX. Uniaxial confined bulk compression was used to determine theparticle strength. Table X lists the results for the protocols describedin Tables II-IX.

TABLE X Results Experiment Table Mass Strength Number referenceDescription change, % change, % 1 II Control −1.3 −22.8 2 III Control;Simulated Saccharide Product Load −0.7 −26.4 3 IV Control; SimulatedAcidic Protein Elution −3.7 −32.9 4 V ISR; low concentration of calciumchloride and buffer 15.7 48.1 5 VI ISR; low concentration of calciumchloride and buffer 13.4 26.5 6 VII Intermittant ISR 3.3 3.9 7 VIII ISR;high concentration of calcium chloride and buffer 16.5 29.3 8 IX ISR;high concentration of calcium chloride and buffer 3.3 5.2

The data for each of the control protocols in experiments 1-3 (i.e.,Tables II-IV) in Table X show little loss in mass and a significantdecrease in particle strength, indicating degradation of the resin.Hydroxyapatite obtained from columns operated using continuousrestoration protocols having low concentrations of calcium chloride andbuffer (i.e., experiments 4 and 5; Tables V and VI) exhibitedsurprisingly significant gains in mass and strength relative to thecontrol protocols, indicating regeneration of the resin. These resultsdemonstrate that the buffered calcium solution provides a significantand surprising degree of regeneration even when a low concentration ofcalcium chloride and buffer is utilized in the restoration protocol.

Hydroxyapatite obtained from columns operated using an intermittentrestoration protocol (i.e., experiment 6; Table VII) had a smallincrease in mass and strength relative to the control protocol,indicating that the intermittent restoration protocol does not degradethe resin. Thus, the hydroxyapatite surprisingly does not requirerestoration after each purification procedure, which can result insavings in process time and cost.

Hydroxyapatite obtained from a column operated for 24 cycles and 1.1column volume using the restoration protocol having a higher calciumchloride and buffer concentration in the buffered calcium solution(i.e., experiment 7, Table VIII) showed an increase in mass and particlestrength compared to the control protocol. These results demonstratethat the use of a buffered calcium solution provides a significant andsurprising degree of regeneration even when a high concentration ofcalcium chloride and buffer is utilized in the restoration protocol.

Hydroxyapatite obtained from a column operated for 5 cycles and 3 columnvolumes using the restoration protocol having a higher calcium chlorideand buffer concentration in the buffered calcium solution (i.e.,experiment 8, Table IX) showed a small gain in mass and particlestrength compared to the control protocol. These results surprisinglyindicate that, when the buffered calcium solution includes a highconcentration of calcium chloride and buffer, fewer cycles and morecolumn volumes of the buffered calcium solution may be utilized.

All patents, patent applications, and other published referencematerials cited in this specification are hereby incorporated herein byreference in their entirety.

What is claimed is:
 1. A method of treating a ceramic hydroxyapatitesolid surface prior to first use in a chromatographic procedure, themethod comprising: (a) providing a ceramic hydroxyapatite solid surface,wherein the ceramic hydroxyapatite surface has not been used in anychromatographic procedure and has not previously been contacted with anysample; (b) contacting the ceramic hydroxyapatite solid surface with aphosphate buffered solution at a pH of at least about 6.5; and then (c)contacting the ceramic hydroxyapatite solid surface obtained in step (b)with a solution having a hydroxide; and then (d) purifying protein froma sample with the ceramic hydroxyapatite solid surface obtained in step(c).
 2. The method of claim 1, wherein the phosphate buffered solutionis a solution having from about 0.1 M to about 1.0 M phosphate at a pHof from about 6.5 to about 10.0.
 3. The method of claim 2, wherein thephosphate buffered solution is 400 mM phosphate at a pH of 8.0.
 4. Themethod of claim 1, wherein the hydroxide is an alkaline metal hydroxide.5. The method of claim 4, wherein the alkaline metal hydroxide is sodiumor potassium hydroxide.
 6. The method of claim 1, wherein the purifyingof step (d) comprises contacting the ceramic hydroxyapatite solidsurface with the sample, thereby separating the protein from one or morebiological compounds; then (e) collecting the protein from the ceramichydroxyapatite solid surface; and then (f) regenerating the ceramichydroxyapatite solid surface, the regenerating comprising, (i)contacting the ceramic hydroxyapatite solid surface with a bufferedcalcium solution having a calcium ion at a concentration of at leastabout 5 mM and a zwitterionic buffer, wherein the ratio of zwitterionicbuffer concentration to calcium ion concentration is at least about 2,and the pH of the solution is at least about 6.5; (ii) contacting theceramic hydroxyapatite solid surface with a phosphate buffered solutionat a pH of at least about 6.5; and (iii) contacting the ceramichydroxyapatite solid surface with a solution having an hydroxide.
 7. Themethod of claim 6, wherein (d) comprises binding the protein to theceramic hydroxyapatite solid surface, and (e) comprises eluting theprotein from the ceramic hydroxyapatite solid surface.
 8. The method ofclaim 6, wherein (d) comprises contacting the ceramic hydroxyapatitesolid surface to the protein, thereby flowing the protein through theceramic hydroxyapatite solid surface, and (e) comprises collecting theprotein in the flow through.
 9. The method of claim 6, wherein thezwitterionic buffer is a sulfonic acid containing buffer.
 10. The methodof claim 9, wherein the sulfonic acid containing buffer is MES, PIPES,ACES, MOPSO, MOPS, BES, TES, HEPES, DIPSO, TAPS, TAPSO, POPSO, orHEPPSO, EPPS, CAPS, CAPSO, or CHES.
 11. The method of claim 9, whereinthe sulfonic acid containing buffer is MES.
 12. The method of claim 6,wherein the calcium ion is at least about 25 mM or at least about 50 mM.13. The method of claim 6, wherein the ratio of zwitterionic bufferconcentration to calcium ion concentration is at least about 2.5. 14.The method of claim 6, wherein the buffered calcium solution comprisescalcium chloride or calcium nitrate.
 15. The method of claim 6, whereinthe phosphate buffered solution comprises a solution containing fromabout 0.1 M to about 1.0 M phosphate at a pH of from about 6.5 to about8.
 16. The method of claim 15, wherein the phosphate buffered solutioncomprises 400 mM phosphate at a pH of 7.0.
 17. The method of claim 6,wherein the regenerating step reverses or eliminates degradation of acolumn that occurs during protein purification or column cleaning steps.18. The method of claim 6, wherein the regenerating step is performedbefore a phosphate cleaning/stripping step.
 19. The method of claim 7,wherein the regenerating step is performed after elution of the protein.20. The method of claim 6, wherein step (f) (ii) further comprises:contacting the ceramic hydroxyapatite solid surface with a solutioncomprising phosphate at a concentration of 10 mM, or less than about 10mM, at a pH of at least about 6.5; and then contacting the ceramichydroxyapatite solid surface with a solution comprising phosphate at aconcentration of at least about 100 mM at a pH of at least about 6.5.21. The method of claim 6, wherein the regenerating step consists of(i), a wash, (ii), and (iii).
 22. The method of claim 1, wherein theprotein is an antibody.